Equipment lubricating microbial compositions

ABSTRACT

An exemplary embodiment is directed to an equipment lubricating composition comprising useful microorganisms. An exemplary embodiment comprises a water insoluble, water-absorbent substance and an encapsulated microorganism component including viable microorganisms. This encapsulating material may encapsulate and protect the microorganisms by essentially preventing the microorganisms from contacting the external environment. Based on the protection afforded by the encapsulation, exemplary embodiments may include previously inhospitable carrier compounds such as particulate machine lubricants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.11/517,051 filed Sep. 7, 2006, which claims the benefit of U.S.Provisional Application Ser. No. 60/715,076 filed Sep. 8, 2005. All ofthe referenced applications, along with International Application No.PCT/US2006/034744, are hereby expressly incorporated by reference intheir entirety.

TECHNICAL FIELD

The disclosed embodiments relate to formulations of viablemicroorganisms for industrial and agricultural applications.

BACKGROUND

Certain microorganisms are produced in large quantities and can beformulated for various commercial uses. For example, microbial productshave been used in agriculture to protect plants from pests and diseases,to improve plant performance and nutrition, and as inoculants forsilages. These microbial products must be produced in a way that isefficient, free of contamination, and suitable for maintaining highlevels of viable microorganisms. Production of microbial formulationsfor commercial use requires drying the microorganisms in a way thatpreserves viability of the microbes, provides a suitable medium forcommercial use, and maintains an extended shelf life of the microbialproduct.

A range of microorganisms have been produced and formulated forcommercial use. Examples of commercially formulated microorganismsinclude strains of Lactobacillus spp. for a variety of food, probiotic,and animal feed uses; entomophagous fungi, such as Beaveria andMetarhizum spp., for control of plant-attacking insects; fungi thatprotect plants from diseases, such as Trichoderma and Clonostachys spp.;bacteria that protect plants from disease, such as Pseudomonas andBacillus spp., as well as Rhizobium and Bradyrhizobium; and relatedbacteria that fix nitrogen through a symbiotic relationship with legumesand fungi, such as Colletotrichum spp., which are used as weed controlsby causing disease in weeds.

Peat-based inoculants presently constitute the vast majority ofinoculants marketed today, and their development is primarily due totheir convenience in holding and distributing desired microorganisms. Inthe known art, such soil-like compositions are required in order toprovide a substrate and a food source for the microorganisms in theinterim period before the microbial products are applied to suchmaterials as seed or plants. For example, in order to maintain highlevels of viable microorganisms, useful microbial products relating toinoculants such as Rhizobium have typically been packaged in a peatmedium or other humus-type carrier.

Many times peat requires processing before it may serve as a carriermedium for desired microorganisms. For example, at least one U.S. patentdescribes a typical process in which pH adjusted sedge peat is ovendried and milled in a hammer mill before being passed through a sieve.The powdered peat is sealed into polyethene bags and sterilized by gammaradiation. All of this occurs before sterilized packs are then injectedwith the desired microorganisms. Post injection, the injection hole isthen re-sealed to prevent contamination.

The use of peat or other humus-type materials is problematic as acarrier medium. First, peat and many other humus-type carrier materialsin the known art are abrasive. Planting equipment and many other typesof equipment may be sensitive to the added friction introduced by thepresence of peat in the formulation. Therefore, when peat basedinoculants are applied to the seed, these formulations may actuallyincrease the friction in the planting equipment (or other industrialequipment which may be used). Peat-based inoculants may tend to increaseseed binding and bridging in planter hoppers, and additionally, mayincrease the wear and tear on critical metering parts and equipment.

Although humus-type materials can sustain desired microbes for extendedperiods, these compositions may be equally suitable for promoting thegrowth of contaminate species. These species may negatively affect theperformance of the desired microorganisms. Although sterilizing the peatmay provide a contaminate-free starting point, contaminate species mayultimately infiltrate and affect the performance of the inoculants.

Accordingly, new and improved methods of producing microbial productsthat are machine friendly, resistant to contaminate microorganisms, andwhich continue to have high activity levels and an extended shelf lifeare needed. Exemplary embodiments are directed to overcoming these andother limitations in the art.

SUMMARY

This and other unmet needs of the prior art are met by embodiedcompositions and methods as described in more detail below. An exemplaryembodiment disclosed herein is directed to an equipment lubricatingcomposition comprising useful microorganisms. An exemplary embodimentcomprises a water insoluble, water-absorbent substance and anencapsulated microorganism component including viable microorganisms.This encapsulating material may encapsulate and protect themicroorganisms by essentially preventing the microorganisms fromcontacting the external environment. Based on the protection afforded bythe encapsulation, exemplary embodiments may include previouslyinhospitable carrier compounds such as particulate machine lubricants.

In a preferred embodiment, the microorganisms (or propagules) will bepresent in sufficient numbers and with sufficient activity to beeffective for a particular agricultural or industrial application.Preferably, the microorganisms may be present in the formulation in anamount of at least about 5×10⁸ colony forming units (“cfu”) per gram offormulation. Finally, an exemplary embodiment may comprise a machinelubricant carrier component. In a preferred embodiment the lubricantcomprises at least one of talc and graphite. For example, machinelubricants such as talc and/or graphite may comprise between about 5-95%of the entire equipment lubricating composition.

Exemplary embodiments also relate to a method of producing equipmentlubricating compositions. An exemplary embodiment may include the stepsof providing an aqueous suspension of viable microorganisms; contactingthe aqueous suspension with an encapsulating material wherein saidencapsulating material is capable of encapsulating the microorganisms;combining the aqueous suspension with a water insoluble, water-absorbentsubstance under conditions effective to produce a formulation of viableencapsulated microorganisms; and combining the encapsulatedmicroorganisms with a particulate machine lubricant. In a preferredmethod, the particulate machine lubricant may comprise talc and orgraphite.

An exemplary embodiment relates to a method for providing agriculturallyuseful microorganisms in a lubricating carrier substrate in a stableproduct form. For example. exemplary embodiments may be stable forpackaging and shelving for extended periods at normal ambienttemperatures. Furthermore, because exemplary embodiments have low wateractivities, they are resistant to contaminating microorganisms.

At least one exemplary embodiment relates to method of treating a plantor a plant seed with an equipment lubricating composition. This methodinvolves providing the equipment lubricating composition as describedsupra and applying the preparation or the formulation to a plant orplant seeds under conditions effective to treat the plant or plant seed.Because these compositions comprise helpful lubricants, they willmitigate the friction caused by processing the microbial products andthe materials contacted by those products in agricultural or industrialequipment such as, for example, planting equipment.

An exemplary embodiment describes a simple process of drying andformulating fungal and bacterial microorganisms that is inexpensive,requires little equipment, and provides products with excellentviability. An exemplary embodiment is free of peat or abrasive materialsthat contribute to the friction of processing the microbial product.Furthermore, because the example compositions may be co-formulated witha machine lubricant medium, they may actually mitigate the frictioncaused by processing the materials that may be contacted by themicrobial products.

The formulations of exemplary embodiments possess sufficient activity ofthe microorganism to be effective in a variety of applications,including applications that require suspension in water, such as spray,drip irrigation, and other water-based deliver system applications. Theformulations of the exemplary embodiments are non-dusty and have a highlevel of cosmetic appeal. The shelf life of the formulations of anexemplary embodiment is significantly longer than the shelf life ofcurrent formulations.

Formulations of an exemplary embodiment may also protect desiredmicroorganisms from the toxic effects of other biological agents orchemicals (e.g., pesticides), providing the opportunity to produceco-formulations of these materials. In addition, the formulations of anexemplary embodiment comprise a barrier to prevent growth of undesirablemicroorganisms which increases the biological efficacy of the desiredmicroorganism. The method of an exemplary embodiment reduces the amountof downstream processing and, therefore, minimizes the amount of damagethat delicate microbial cells or spores may sustain.

DESCRIPTION

U.S. patent application Ser. No. 11/517,051, fully incorporated byreference in its entirety, discloses formulations for microorganisms.That application also discloses methods for producing and utilizingthose formulations. Exemplary embodiments described herein utilize themicrobial products and methods described in application Ser. No.11/517,051, particularly those directed toward encapsulatedmicroorganisms, along with other substances to produce new and usefulcompositions and methods. Exemplary embodiments described herein aredirected at new and useful compositions made possible because of theprotection afforded by encapsulating desired microorganisms.

An exemplary embodiment includes an equipment lubricating compositionincluding a water insoluble, water-absorbent substance and anencapsulated microorganism component. The encapsulated microorganismcomponent may include an effective amount of viable microorganisms andan encapsulating material which is capable of encapsulating themicroorganisms under appropriate conditions. Remarkably, exemplaryembodiments may also be co-formulated with significant quantities ofmachine lubricants. For example, the equipment lubricating compositionsmay include a particulate machine lubricant such as talc and orgraphite. In an exemplary embodiment, the particulate machine componentmay comprise about 5-95% of the weight of the final composition.Furthermore, exemplary embodiments do not require the presence of peator other soil-like adjuvant.

Even with the presence of significant amounts of machine lubricant, theencapsulated microorganism component of an exemplary embodiment maycomprise an effective amount of viable microorganisms to inoculate atarget material (e.g., seed, plant, or other material capable of beinginoculated). The effective amount of viable microorganisms necessary toinoculate a given target material may vary considerably depending on thedesired microorganism and the intended application. For someapplications, an effective amount of viable microorganisms will be atleast about 5×10⁶ cfu per gram of the formulation. In otherapplications, an effective amount will be at least about 5×10⁸ cfu pergram of the formulation.

Specific exemplary embodiments relate to a method of producing equipmentlubricating compositions. An exemplary embodiment may include the stepsof providing an aqueous suspension of viable microorganisms; contactingthe aqueous suspension with an encapsulating material wherein saidencapsulating material is capable of encapsulating the microorganisms;combining the aqueous suspension with a water insoluble, water-absorbentsubstance under conditions effective to produce a formulation of viableencapsulated microorganisms; and combining the encapsulatedmicroorganisms with a particulate machine lubricant. In a preferredmethod, the particulate machine lubricant may comprise talc and orgraphite.

Combining the aqueous suspension of viable microorganisms with the waterinsoluble, water-absorbent substance can be carried out by a variety ofmethods. In a preferred embodiment, the combining step is carried out bykneading the aqueous suspension with the water insoluble, waterabsorbent substance.

Exemplary methods may use viable microorganisms provided in an aqueoussuspension, or an otherwise suitable environment for maintaining theviability of the microorganisms. The aqueous suspension of viablemicroorganisms may then be contacted with an encapsulating material.When the aqueous suspension of viable microorganisms containing anencapsulating material is combined with a water insoluble,water-absorbent substance, the viable microorganisms may beencapsulated. Preferably, the encapsulation may occur as the waterinsoluble, water-absorbent substance absorbs water from the suspensioncontaining the encapsulating material. As the water insoluble, waterabsorbent substance begins to sequester the fluid, the encapsulatingmaterial may form microbeads or capsules which may provide a relativezone of protection for the microorganisms so encapsulated.

In an exemplary embodiment, the water insoluble, water-absorbentsubstance can be any organic or inorganic material capable of removingmoisture gently from the suspension of viable microorganisms. Suitableinorganic substances include zeolite, porous beads or powders, silica,and the like. Suitable organic substances include plant materials, suchas ground agricultural products (e.g., corn cobs), porous wood products,cellulose, and the like. In addition, cyclodextrins may be useful aswater insoluble, water-absorbent substances. Cyclodextrins are widelyused in many industries for encapsulation/binding of a wide variety ofrelatively apolar materials.

Cyclodextrins are inexpensive, available in bulk quantities, and havelow solubility in water (depending upon the actual composition of thecyclodextrin molecule). Cyclodextrins can be used as formulating agentsto sequester liquid or apolar solid materials that can be suspended inwater, such as pesticides and pesticide adjuvant. Any combination of theabove-described water insoluble, water-absorbent substances may also beused. In a preferred embodiment, the water insoluble, water-absorbentsubstance is a finely ground cellulose powder.

The water insoluble, water-absorbent substance is combined with theaqueous suspension of viable microorganisms to an amount of about 80 to99% by total weight of the pre-lubricant formulation. Thus, the aqueoussuspension of viable microorganism is present in the formulation in anamount of about 1 to 20% by total weight of the formulation. Preferably,the water insoluble, water-absorbent substance is present in theformulation in the amount of at least about 80%, 85%, 90%, 95%, or 99%by total weight of the pre-lubricant formulation.

In an exemplary embodiment, the encapsulating material is a watersoluble material capable of forming a film or microbead when dried.Suitable encapsulating materials include, without limitation, native ormodified chitosans, native or modified starches, glucans or dextrins,celluloses modified so they are soluble, and any of a number of nativeor modified vegetable or microbial gums, including agars, guar, locust,carrageenan, xanthans, pectins, and the like, and combinations thereof.In at least one exemplary embodiment, the encapsulating material is adextrin, such as Crystal-Tex (National Starch and Chemical Co.,Bridgewater, N.J.).

Encapsulating the microorganisms provides many advantages. Inparticular, due to the protection afforded by encapsulation, anexemplary embodiment may comprise a machine lubricant carrier component.In a preferred embodiment the lubricant may comprises at least one oftalc and graphite. For example, machine lubricants such as talc and/orgraphite may comprise between about 5-95% of the entire equipmentlubricating composition.

Machine lubricating powders such as talc and graphite are normallyinhospitable to most microorganisms. However, because the encapsulatedmicroorganisms are essentially isolated from external contact, theencapsulated microorganisms may be co-formulated with essentially drymachine lubricants such as talc and graphite. Various other particulatemachine lubricants may also be used with acceptable results. Forexample, granulated Teflon or other materials with similar propertiesmay be appropriate for particular applications.

Encapsulated microorganisms may be more resistant to chemicalpesticides, which may dramatically reduce the shelf life ofnon-encapsulated microorganisms by contact toxicity. For example, it maybe desirable to combine encapsulated microorganisms with other chemicalor biological agents. When the microorganisms are placed together in acontainer with e.g., a chemical fungicide, the chemical fungicide wouldbe detrimental to a non-encapsulated microorganism. Thus, encapsulationprevents contact of the microorganisms with the chemical pesticide,unless the chemical pesticide has a significant vapor pressure.Encapsulation of the microorganisms may, therefore, be advantageous whenformulating mixtures of microorganisms with chemical pesticides andother biological products.

In exemplary embodiments, encapsulation provides an opportunity todefine a targeted environment for the desired microorganisms. Themicrobeads or capsules that encapsulate the useful microorganisms can bemanipulated to increase the survival and activity of the microorganismsthey encapsulate. For example, nutritional supplements that favor thedesired microorganism may be added to the pre-encapsulation suspension.In some embodiments, the encapsulation material itself may be selectedto provide a food source for the encapsulated microorganisms.

Exemplary embodiments disclosed herein may not only eliminate therequirement for peat or other peat-like materials, they also containlubricants designed to lubricate the machinery and equipment which maybe used to process the desired seeds or plants. Based on the protectiveproperties afforded by the microbeads that encapsulate the desiredmicroorganisms, the target microorganisms may be packaged with normallyinhospitable compounds. Furthermore, the encapsulated microorganisms donot require peat or any other material to sustain the activity orsurvival of the microorganisms.

Proper lubrication is an important aspect of essentially all knownmachines. However, many microorganisms useful for industrial andagricultural purposes cannot survive for extended periods in thepresence of dry machine lubricants such as talc and or graphite. First,the lubricants, such as talc or graphite, are not a food source for themicroorganisms. Accordingly, if packaged without peat or similar lifesustaining materials, most microorganisms will simply run out of food.Second, dry machine lubricants do not retain moisture. Therefore, themicroorganisms will desiccate before they can be used in the relevantapplication. Therefore, the known art has been unable to package manyuseful microorganisms in a lubricant carrier without includingsignificant amounts of a carrier compound such as peat.

Equipment lubricating compositions of viable microorganisms produced bythe methods of the exemplary embodiments may be dried. However, dryingof the microorganisms is preferably performed using methods thatmaintain the viability of the microorganisms. Drying may be performedbefore or after the machine lubricant is combined with the rest of thecomposition. In a preferred embodiment, the drying method is air drying.Air drying is a method that prevents the microorganisms from coming intocontact with high temperatures. Drying the compositions may increase theshelf-life of exemplary embodiments.

The encapsulated microorganism component of exemplary embodiments may bedissolved when appropriate. Accordingly, a further aspect of anexemplary embodiment relates to a preparation of viable microorganismsincluding dissolving the soluble components of a disclosed embodiment insolution. The encapsulated microorganism component of exemplaryembodiments may be suspended in a solution for commercial applicationsin a spray, drip irrigation, or other water-based delivery system.

However, this list of equipment and processes that may benefit fromexemplary embodiments is not intended to be limiting. With appropriatemeasures well known to those of skill in the art, products and methodsdescribed herein may be used with many other mechanical deliverysystems. Exemplary embodiments may be designed according to the scaleand needs of a particular application.

Another aspect of an exemplary embodiment relates to method of treatinga plant or a plant seed with an equipment lubricating compositioncontaining desired microorganisms. This method involves providing apreparation or a formulation as described supra and applying thepreparation or the formulation to a plant or plant seed under conditionsand in an amount effective to treat the plant or plant seed. Because anexemplary embodiment includes a particulate machine lubricant, theapplication of the preparation to the plant or plant seeds may reducethe friction associated with processing the desired microorganismsthemselves or the treated plant or plant seed. In an exemplaryembodiment, the particulate machine lubricant will not dissolve and willbe dispersed on the plant or plant seed.

Treating a plant or a plant seed according to the method of an exemplaryembodiment may include, without limitation, imparting diseaseresistance, imparting resistance to pests, improving nutrition and/oryield, or any combination thereof.

In addition to the microorganisms themselves, propagules of themicroorganisms may also be formulated pursuant to the methods of anexemplary embodiment. Propagules include, without limitation, fungalspores, hyphae, vesicles, and auxiliary cells. The microorganisms mayhave a commercial utility in agricultural applications and may be usefulto control insect pests, weeds, and plant disease, or to providenourishment to plants so that their growth and/or yield is increased.Suitable species of microorganisms include, without limitation, speciesof the genera Bacillus and Pseudomonas (useful for insect control);Beauveria, Metarhizum, and species of the division Oomycota (weedcontrol); Colletotrichum, Phytophthora, Trichoderma, Clonostachys, andbinucleate Rhizoctonia (to control plant diseases); and Bradyrhizobium,Rhizobium and related genera (to improve plant nutrition and yieldincluding). Other similar microorganisms can also be formulated usingthe method of an exemplary embodiment. Microorganisms with commercialutility in food processing, brewing, and silage and sewage treatment arealso suitable for formulation pursuant to the method of an exemplaryembodiment. Preferred microorganisms may include species of the fungusTrichoderma and species of the bacteria Bradyrhizobium.

These and other aspects of the exemplary embodiments are furtherillustrated by the examples below.

EXAMPLES Example 1 Preparation of Equipment Lubricating Compositions

To prepare the encapsulated microorganism component, a frozen paste ofconcentrate Bradyrhizobium from a commercial source was used as astarting material. The initial Bradyrhizobium suspension contained about1×10¹² colony forming units/ml of the bacterium. Crystal Tex dextrin wasadded to the suspension to give a 4% w/v mixture and mixed until it wasdissolved. Cellulose (Sigmacell Type 50, Sigma Chemical Co., St. Louis,Mo.) was added to each preparation to give two volumes of cellulose foreach volume of cell suspension+Crystal Tex. These were mixed by kneadingby hand in a plastic bag until a uniform mixture was obtained. Theresult was a free-flowing powder. This material was air dried until thewater activity was approximately 0.55. This material constitutes anexample encapsulated microorganism component.

A particulate machine lubricant comprising 18.8 ounces of talc and 4.7ounces of graphite was combined with 1.5 ounces of the encapsulatedmicroorganism component above. The package size of the finished productwas 25 ounces.

Example 2 Characterization of the Product Composition

Sterile PBS was added to the preparation from Example 1 to dissolve theencapsulated microorganism component. A standard dilution platingprotocol was performed to determine the cfu levels and stability of thegraphite, talc, and encapsulated microorganisms in a finishedformulation. The cfu counts of Bradyrhizobia in the final compositionwere determined to be 4×10⁹ cfu/g.

The description of the disclosed embodiments is presented for purposesof clarity of understanding only, and no unnecessary limitations shouldbe implied. It will be appreciated by those skilled in the art thatembodiments may be practiced in various alternate forms andconfigurations. For example, although many of the examples discussed aredirected to microorganisms useful in an agricultural context, it shouldbe appreciated that the invention is not so limited. The conceptexemplified in the example embodiments may apply broadly to othersituations in which desired microbial products or the materials theytreat may contact or be processed by machinery. Likewise, despite thefact that particular microorganisms are discussed in more detail thanothers, many other useful microorganisms which are not specificallymentioned may also be encapsulated and formulated pursuant to thecompositions and methods exemplified by the disclosed embodiments.Microorganisms may include, without limitation, species from thekingdoms Eubacteria, Archaebacteria, Protista, and Fungi.

1. An equipment lubricating composition, comprising: a water insoluble,water-absorbent substance; an encapsulated microorganism componentincluding: an effective amount of viable microorganisms to inoculate;and an encapsulating material which encapsulates the microorganisms; anda particulate machine lubricant including at least one of talc andgraphite.
 2. The composition of claim 1, wherein the particulate machinelubricant comprises about 5-95% by weight of the equipment lubricatingcomposition.
 3. The composition according to claim 1, wherein a waterinsoluble, water-absorbent substance, wherein the water insoluble,water-absorbent substance is present in an amount of at least about 90%by total weight of the formulation and microorganisms mixed with thewater
 4. The formulation according to claim 2, wherein the waterinsoluble, water-absorbent substance is selected from the groupconsisting of zeolite, porous beads or powders, silica, groundagricultural products, porous wood products, cellulose, cyclodextrins,and combinations thereof.
 5. The formulation according to claim 2,wherein the water insoluble, water-absorbent substance is cellulosepowder.
 6. The formulation according to claim 2, wherein themicroorganisms are selected from the group consisting of fungi,bacteria, and combinations thereof.
 7. The formulation according toclaim 6, wherein the microorganisms are fungi.
 8. The formulationaccording to claim 7, wherein the fungi is a species from the genusTrichoderma.
 9. The formulation according to claim 6, wherein themicroorganisms are bacteria.
 10. The formulation according to claim 9,wherein the bacteria is a species from the genus Bradyrhizobium.
 11. Apreparation of viable microorganisms comprising the formulation of claim1 wherein the encapsulated microorganism component is dissolved insolution.
 12. A method of treating a plant or a plant seed with anequipment lubricating composition, said method comprising: providing apreparation according to claim 11 and applying the preparation to aplant or plant seed under conditions effective to treat the plant orplant seed.
 13. The method according to claim 12, wherein theparticulate machine lubricant is dispersed on the plant or plant seed.14. A method of treating a plant or a plant seed with a microorganism,said method comprising: providing a formulation according to claim 2 andapplying the formulation to a plant or plant seed under conditionseffective to treat the plant or plant seed.
 15. A method of producing anequipment lubricating composition, said method comprising: providing anaqueous suspension of viable microorganisms; contacting the aqueoussuspension with an encapsulating material wherein said encapsulatingmaterial is capable of encapsulating the microorganisms; combining theaqueous suspension with a water insoluble, water-absorbent substanceunder conditions effective to result in viable encapsulatedmicroorganisms; and combining the encapsulated microorganisms with aparticulate machine lubricant.
 16. The method according to claim 15,wherein the particulate machine lubricate comprises at least one of talcand graphite.
 17. The method according to claim 16, wherein saidcombining step is carried out by kneading the aqueous suspension withthe water insoluble, water-absorbent substance.
 18. The method accordingto claim 15, further comprising: drying the encapsulated microorganismsunder conditions effective to maintain the viability of themicroorganisms; and
 19. The method according to claim 16, furthercomprising: drying the encapsulated microorganisms with air underconditions effective to maintain the viability of the microorganisms.